Part:BBa_K2305018

GFP with pcat

Pcat+RBS+GFP+T

The purpose of this biobrick is to produce as much green fluorescence as possible, and ensure a strong GFP expression in E.coli.

This biobrick is an improvment of the biobrick BBa_K5876070 designed by iGEM12_UT_Dallas.

Characterizing Part:

BBa_K2305018 through measuring green fluorescence intensity in E.coli Trans5α and comparing the promoter strength with mutants.(Fig.1)

Fig.1

Whether eukaryotes or prokaryotes, each species exhibits some degree of difference or preference for codon usage. These preferences may be related to two reasons: one is to avoid the use of similar termination codon; another is able to translate these preferences codon effectively, because these codons correspond to very rich tRNA in living organisms. Whatever the cause of this preference, the bias of codon usage among different organisms can be very large. Therefore, we do protein expression or production, we need to consider the problem of codon bias. By using the preferred codons, and avoiding the low or rare codons, the redesign of the gene expressed in the target is also called codon optimization.

We used the microplate reader to compare their OD value and the fluorescence value under the same experimental conditions (Fig.2;Fig.3). As the figure demonstrates, in the first eight hours the OD600 value increased gradually, the BBa_K2305018 expressed high level expression in E.coli than the previous part. It can be an expression part is improved over the initial design.

Fig.2

Fig.3

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 708